This application addresses broad Challenge Area (04): Clinical Research, and specific topic: 04-AR-106 Cellular, Molecular and Genetic Therapies for Rare Inherited Diseases of Muscle, Skin and Connective Tissue and Bone. Duchenne Muscular Dystrophy (DMD) is the most common lethal genetic disease of childhood, occurring in 1 in every 3500 live male births. Based on the structure of dystrophin, as well as the mutational profile of patients with DMD, this disease is potentially amenable to an exon skipping therapeutic strategy for the majority of those affected. Most of the mutations in DMD result from DNA deletions between exons 44-55. Such deletions usually lead to out of frame transcripts, which result in lack of dystrophin protein production. Recent strategies aimed at anti-sense oligonucleotide (AON) directed removal of specific exons during processing of the dystrophin transcript have succeeded in restoring reading frame to a fraction of the transcripts, leading to some production of partially functional dystrophin protein. While early stage clinical trials are underway, the ultimate success of this therapeutic approach rests on overcoming the inefficiencies of exon skipping from systemically administered AON. Best estimates indicate that 30-60% of wild-type levels of (skipped) dystrophin will be required to functionally compensate for loss of dystrophin at a significant level. Early trial data, while promising, indicate that even local IM delivery of AON falls short of inducing such levels, yielding only 3-35% of normal dystrophin amount. It is anticipated that systemic delivery of AON may be even more inefficient. Therefore, identification of compounds that increase the efficacy of exon skipping represents a viable approach toward increasing replacement of dystrophin to functionally relevant levels. We have implemented high throughput screens to identify small molecule compounds capable of perturbing the splicing machinery to favor exonic exclusion in the context of targeted AON. The program has identified 20 compounds, most of which are already FDA approved drugs that now need to be assessed in the context of various relevant human mutations and in mouse models. Here we set up a multi-PI program to bring together experts in cell biology, genomics, and muscular dystrophy to identify drugs for use as adjuvants to AON-mediated exon skipping clinical trials. Ultimately, the program will include the search for novel structures and medicinal chemistry, but this is outside the scope of this proposal, which is intended to be completed within two years. Thus, the project is highly responsive to the Challenge Topic, which states "novel therapeutic approaches offer the possibility of restoring function to a defective gene or compensating for the loss of gene function. These approaches are potentially quite powerful and could lead to significant advances in the treatment of diseases of muscle and other tissues. The goal of the projects will be to find creative approaches to overcome some of the current technical obstacles.... Areas of interest include ..., methods for editing gene products in vivo, such as exon-skipping antisense oligonucleotides and small RNAs." Here we propose to create immortalized DMD patient derived fibroblasts, which are readily and reproducibly inducible to myotubes and to develop quantitative methods for detecting mutant and skipped DMD mRNA products in these cells. Once developed, 20 lead compounds will be screened for their efficacy and specificity in facilitating AON exon skipping. Assessment of the activity of these same compounds on myoblast/myotube cultures from mouse DMD models mdx and mdx.4Cv will enable a further assessment of specificity and identify candidates which we will test in the mdx or mdx.4Cv models in vivo. The value of such an approach is threefold: 1) Creation, development and immortalization of patient derived cells inducible to muscle lineage will create a much needed resource that can be distributed and utilized by muscular dystrophy researchers for preclinical assessment of multiple potential therapeutics. Because many emerging treatments are specific to human muscular dystrophy mutations, availability of preclinical assessment tools based in human cells with relevant mutations are lacking, and represent a technical and ethical barrier toward moving clinical trials forward. 2) Screening lead compounds on these cells alongside mouse myoblast/myotubes and in mouse models in vivo will enable us to validate the predictive value of the in vitro assays on in vivo outcome in DMD models. Thus, we will have created a process for screening and validating compounds emerging from larger screens or Structure Activity Relationship analysis (SAR). 3) Implementing these screens on 20 lead compounds has the potential to validate their activity, determine their specificity, and identify potential target DMD populations. Identification of a compound that can improve efficacy of AON directed exon skipping has the potential to move this therapeutic modality from proof of principle of dystrophin production to therapeutic efficacy resulting in functional improvement, thus rendering exon skipping a practical and effective treatment for DMD. While DMD is the target in this application, we note that the methods and the approach that we are taking will make the compounds identified generally useful in other disorders amenable to an exon skipping strategy. Exon skipping is a promising emerging therapy for Duchene Muscular Dystrophy (DMD), the most common lethal genetic disease of childhood. Early clinical trial results predict that this approach can restore the missing dystrophin protein to muscle in DMD patients, but at levels insufficient to result in functional gain. Identification of compounds that can improve the efficacy of exon skipping has the potential to move this therapeutic modality from proof of principle to therapeutic efficacy, rendering exon skipping a practical and effective treatment for DMD.